Design and characterization of influenza vaccines

ABSTRACT

Provided herein are optimized, recombinant influenza HA polypeptides that elicit immune responses. Also provided are compositions and kits comprising the optimized HA polypeptides as well as methods of making and using the optimized HA polypeptides.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/221,336, filed Sep. 21, 2015, which is incorporated herein by reference in its entirety.

BACKGROUND

Influenza virus is a member of Orthomyxoviridae family. There are three subtypes of influenza viruses, designated influenza A, influenza B, and influenza C. The influenza virus contains a segmented negative-sense RNA genome, which encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). The HA, NA, M1, and M2 are membrane associated, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsid associated proteins. The HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell, and the sources of the major immunodominant epitopes for virus neutralization and protective immunity. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA. These different types of HA and NA form the basis of the H and N distinctions in, for example, H5N1. There are 16 H and 9 N subtypes known, but only H 1, 2 and 3, and N 1 and 2 are commonly found in humans. Both HA and NA proteins are considered the most important components for prophylactic influenza vaccines.

BRIEF SUMMARY

Provided herein are optimized, recombinant influenza HA polypeptides that elicit immune responses. Also provided are compositions and kits comprising the optimized HA polypeptides as well as methods of making and using the optimized HA polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing expression of exemplary optimized HA polypeptides in VLP platform at 72 hours.

FIG. 2 is a schematic of the results of a hemagglutination inhibition (HAI) assay. Following vaccination in mice, serum samples were collected at week 10 post-vaccination and sera was tested in an HAI assay against a panel of wild-type H3N2 influenza viruses. The number of mice (out of 5 mice per group) that reached the 1:40 threshold is listed as the number in the table.

FIG. 3 are graphs showing HAI titers elicited by wild-type H3 HA VLP vaccines and T-6 and T-7 COBRA vaccines.

FIG. 4 is a schematic showing the geometric mean HAI titers and number of strains recognized by elicited antisera against the 10 historical wild-type H3N2 strains.

FIG. 5 is a schematic showing the geometric mean HAI titers and number of strains recognized by elicited antisera against the H3N2 dirft varaint strains (2004-2007).

FIG. 6 is a graph showing the strains with HAI reactivity against a panel of 15 2004-2009 virus variants.

DETAILED DESCRIPTION

Current vaccination approaches primarily rely on the induction of antibodies recognizing the hemagglutinin (HA) protein. The HA glycoprotein is expressed as a trimeric complex of identical subunits on the surface of influenza virions, and mediates virus attachment and subsequent membrane fusion with target cells (Skehel and Wiley, Annu. Rev. Biochem. 69:531-569 (2000); Bouvier and Palese, Vaccine 12:26, Suppl. 4:D49-53 (2008), which are incorporated by reference herein in their entireties). Individual HA monomers can be further segregated into the membrane distal globular head and membrane proximal stalk domains. The globular head encodes the receptor-binding site (RBS) and the stalk domain encodes the fusion peptide.

Antibodies directed against HA, and more specifically to epitopes in close proximity to the RBS within the globular head region, are elicited following influenza infection or vaccination (Gerhard et al., Nature 290(5808):713-7 (1981); Wilson et al., Virology 458-459:114-24 (2014); Carter, et al., J. Virology, 87(3):1400-10 (2013); Wrammert, et al., Nature, 453(7195):667-71 (2008); Huang, et al., J. Infect. Dis., 209(9):1354-1361 (2014), which are incorporated by reference herein in their entireties). These antibodies possess potent neutralization capacity through their ability to interfere with viral attachment to target cells and are readily detected using the hemagglutinin inhibition (HAI) assay (Skehel and Wiley, Annu. Rev. Biochem. 69:531-569 (2000); Ohmit, et al., J. Infect. Dis. 204(12):1879-85 (2011), which are incorporated by reference herein in their entireties). While antibodies with HAI activity can prevent influenza infection, they are largely strain-specific. Accumulation of point mutations within the globular head region of HA, termed antigenic drift, generates viral escape variants and often evasion of pre-existing immunity (Ohmit, et al., J. Infect. Dis. 204(12):1879-85 (2011); Webster, Nature, 296(5853):115-21 (1982); Knossow and Skehel, Immunology 119(1):1-7 (2006), which are incorporated by reference herein in their entireties). Moreover, antigenic drift necessitates frequent reformulation of the seasonal vaccine; a process that is both expensive and time-consuming. Therefore, a need exists for development of vaccines that generate broadly cross-reactive neutralizing antibodies. Such vaccines are provided herein.

Hemagglutinin (HA) is an influenza virus surface glycoprotein. HA mediates binding of the virus particle to a host cells and subsequent entry of the virus into the host cell. The nucleotide and amino acid sequences of numerous influenza HA proteins are known in the art and are publically available, such as those deposited with GenBank. HA (along with NA) is one of the two major influenza virus antigenic determinants. Provided herein are computationally optimized broadly reactive antigen (COBRA) HA proteins. Specifically, provided herein are isolated recombinant influenza HA polypeptides comprising at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. Optionally, the isolated recombinant influenza HA polypeptides comprise SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. Optionally, the isolated recombinant influenza HA polypeptides are SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.

A recombinant nucleic acid, protein or virus is one that has a sequence that is not naturally occurring and/or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. When used in reference to the optimized influenza HA polypeptides describe herein, for example, the term refers to the fact that the HA polypeptides have been designed (i.e., are not naturally occurring) and/or whose existence and production require one or more actions. The artificial production or combination is often accomplished by chemical synthesis and/or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

As used herein, an “isolated” biological component (such as a nucleic acid, protein or virus) has been substantially separated or purified away from other biological components (such as cell debris, or other proteins or nucleic acids). Biological components that have been “isolated” include those components purified by standard purification methods. The term also embraces recombinant nucleic acids, proteins or viruses, as well as chemically synthesized nucleic acids or peptides.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Also provided herein are isolated nucleic acid molecules comprising nucleotide sequences encoding the provided recombinant, hybrid influenza hemagglutinin (HA) polypeptides. Specifically, provided are isolated recombinant nucleic acid molecules comprising a nucleotide sequence encoding an influenza hemagglutinin (HA) polypeptide, wherein the nucleotide sequence encoding the HA polypeptide is at least at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26. Optionally, the nucleic acid sequences comprise SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. The term “nucleic acid” includes single-, double-, or multiple-stranded DNA, RNA and analogs (derivatives) thereof. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. In certain embodiments. the nucleic acids herein contain phosphodiester bonds. In other embodiments, nucleic acid analogs are included that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and nonribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

Nucleic acids are linear polymers (chains) of nucleotides, which consist of a purine or pyrimidine nucleobase or base, a pentose sugar, and a phosphate group. As used herein, a “polymer backbone” refers to the chain of pentose sugars and phosphate groups lacking the bases normally present in a nucleic acid.

Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” refers to a nucleic acid sequence that contains a series of residues that are not complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is incapable of hybridizing to any other nucleic acid sequence under hybridizable conditions. Optionally, a nonspecific nucleic acid sequences is a sequence that is not substantially identical to any other nucleic acid sequence. By way of another example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid. An “inhibitory nucleic acid” is a nucleic acid (e.g. DNA, RNA, polymer of nucleotide analogs) that is capable of binding to a target nucleic acid (e.g. an mRNA translatable into a protein) and reducing transcription of the target nucleic acid (e.g. mRNA from DNA) or reducing the translation of the target nucleic acid (e.g., mRNA) or altering transcript splicing (e.g. single stranded morpholino oligo).

A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

A “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species of group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells. Codon optimization does not alter the amino acid sequence of the encoded protein.

The terms “identical” or percent sequence “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site at ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Employed algorithms can account for gaps and the like.

For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively.

Also provided are vectors comprising nucleic acid sequences encoding the provided influenza hemagglutinin (HA) polypeptides. Specifically, provided are isolated vectors comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26. Also provided are a plurality of vectors comprising (i) a vector comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, encoding an HA polypeptide; (ii) a vector comprising a nucleic acid sequence encoding an influenza neuraminidase (NA); and (iii) a vector comprising a nucleic acid sequence encoding an HIV GAG polypeptide. Optionally, provided are a plurality of vectors comprising (i) a vector comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, encoding an HA polypeptide; (ii) a vector comprising a nucleic acid sequence encoding an influenza neuraminidase (NA); and (iii) a vector comprising a nucleic acid sequence encoding an influenza M1 polypeptide. Optionally, the amino acid sequence of the NA comprises SEQ ID NO:27 and the amino acid sequence of the M1 polypeptide comprises SEQ ID NO:29. Optionally, the nucleic acid sequence encoding the NA polypeptide comprises SEQ ID NO:28 and the nucleic acid sequence encoding the M1 polypeptide comprises SEQ ID NO:30.

As used herein, a matrix (M1) protein refers to the influenza virus structural protein found within the viral envelope. M1 is thought to function in assembly and budding. As used herein, a neuraminidase (NA) refers to the influenza virus membrane glycoprotein. NA is involved in the destruction of the cellular receptor for the viral HA by cleaving terminal sialic acid residues from carbohydrate moieties on the surfaces of infected cells. NA also cleaves sialic acid residues from viral proteins, preventing aggregation of viruses. NA (along with HA) is one of the two major influenza virus antigenic determinants.

As used herein, a vector refers to a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. An insertional vector is capable of inserting itself into a host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. In some embodiments of the present disclosure, the vector encodes an influenza HA, NA or M1 protein. Optionally, the vector is the pTR600 expression vector (U.S. Patent Application Publication No. 2002/0106798; Ross et al., Nat. Immunol. 1(2):102-103, 2000; Green et al., Vaccine 20:242-248, 2001, which are incorporated by reference herein in their entireties).

Construction of suitable vectors containing the nucleic acid sequences employs standard ligation and restriction techniques, which are well understood in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired.

The provided vectors can contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. Enhancers can be used to function to increase transcription from nearby promoters. Vectors may also contain sequences necessary for the termination of transcription referred to as terminators. The identification and use of terminators in expression vectors is well established. Further, the vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed.

Cells comprising the vectors and nucleic acids are provided. Such cells can be used to generate the provided recombinant, hybrid HA polypeptides and virus-like particles containing the recombinant, hybrid, HA polypeptides. Suitable cells include, but are not limited to, eukaryotic host cells, prokaryotic host cells, and mammalian host cells. For example, the cells can be yeast, insect, avian, plant, C. elegans, and mammalian host cells. Examples of mammalian cells include, but are not limited to, COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero, and Hep-2 cells. Prokaryotic host cells include bacterial cells, for example, E. coli, B. subtilis, and mycobacteria.

Provided are methods of making a virus like particle (VLP) comprising culturing the cells comprising the provided nucleic acids or vectors under conditions to produce the VLP and isolating the VLP. Provided is a method of making a VLP comprising transfecting a host cell with a vector encoding a HA polypeptide, a vector encoding an influenza NA polypeptide and a vector encoding an influenza Ml polypeptide under conditions sufficient to allow for expression of the HA, Ml and NA polypeptides and isolating the VLP. Provided is a method of making a VLP comprising transfecting a host cell with a vector encoding an HA polypeptide of, a vector encoding an influenza NA polypeptide and a vector encoding a HIV GAG polypeptide under conditions sufficient to allow for expression of the HA, HIV GAG and NA polypeptides and isolating the VLP. Optionally, the polypeptides comprise a sequence that is at least 95% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. Optionally, the polypeptides comprise SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. Optionally, the nucleic acid sequences comprise a sequence that is at least 95% identical to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26. Optionally, the nucleic acid sequences comprise SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26. Optionally, the amino acid sequence of the NA comprises SEQ ID NO:27 and the amino acid sequence of the M1 polypeptide comprises SEQ ID NO:29. Optionally, the nucleic acid sequence encoding the NA polypeptide comprises SEQ ID NO:28 and the nucleic acid sequence encoding the M1 polypeptide comprises SEQ ID NO:30.

The influenza VLPs are made up of the HA, NA and M1 proteins. The production of influenza VLPs has been described in the art and is within the abilities of one of ordinary skill in the art. As described herein, influenza VLPs can be produced by transfection of host cells with plasmids encoding the HA, NA and M1 proteins. After incubation of the transfected cells for an appropriate time to allow for protein expression (such as for approximately 72 hours), VLPs can be isolated from cell culture supernatants. See, for example, U.S. Pat. No. 8,883,171, which is incorporated herein by reference in its entirety. The influenza VLPs disclosed herein can be used as influenza vaccines to elicit a protective immune response against influenza viruses.

Provided are isolated virus-like particles comprising the provided HA polypeptides. Optionally, the virus-like particles comprise an influenza neuraminidase (NA) polypeptide and an influenza matrix (M1) polypeptide. Optionally, the amino acid sequence of the influenza NA polypeptide is at least 95% identical to SEQ ID NO:27, the amino acid sequence of the influenza M1 polypeptide is at least 95% identical to SEQ ID NO:29, or both. Optionally, the virus-like particles comprise an influenza neuraminidase (NA) polypeptide and an HIV GAG polypeptide.

As used herein, virus-like particles (VLP) refer to virus particles made up of one of more viral structural proteins, but lacking the viral genome. Because VLPs lack a viral genome, they are non-infectious. In addition, VLPs can often be produced by heterologous expression and can be easily purified. Most VLPs comprise at least a viral core protein that drives budding and release of particles from a host cell. One example of such a core protein is influenza M1. In some embodiments herein, an influenza VLP comprises the HA, NA and M1 proteins. As described herein, influenza VLPs can be produced by transfection of host cells with plasmids encoding the HA, NA and M1 proteins. After incubation of the transfected cells for an appropriate time to allow for protein expression (such as for approximately 72 hours), VLPs can be isolated from cell culture supernatants. Example 1 provides an exemplary protocol for purifying influenza VLPs from cell supernatants. In this example, VLPs are isolated by low speed centrifugation (to remove cell debris), vacuum filtration and ultracentrifugation through 20% glycerol.

Optionally, recombinant whole influenza viruses comprising the herein provided VIPER HA polypeptides are produced. Recombinant whole influenza viruses can be produced by plasmid-based reverse genetics and cell-based or egg-based technologies. Recombinant viruses containing internal protein genes from a donor virus may be used to prepare inactivated influenza virus vaccines (see, e.g., Fodor, E. et al. Rescue of influenza A virus from Recombinant DNA. J. Virol., 1999, 73, 9679-9682; incorporated by reference herein). Recombinant whole influenza viruses can be used to elicit protective immune responses against influenza viruses; for example, they can be administered as components of a live-attenuated or split-inactivated vaccine.

Thus, the immunogenic compositions and vaccines described herein may comprise one of three types of antigen preparation: inactivated whole virus, sub-virions where purified virus particles are disrupted with detergents or other reagents to solubilize the lipid envelope (“split” vaccine) or purified structural influenza polypeptide (“subunit” vaccine). Optionally, virus can be inactivated by treatment with formaldehyde, beta-propiolactone, ether, ether with detergent (such as TWEEN-80®), cetyl trimethyl ammonium bromide (CTAB) and Triton N101, sodium deoxycholate and tri(n-butyl) phosphate. Inactivation can occur after or prior to clarification of allantoic fluid (from virus produced in eggs); the virions are isolated and purified by centrifugation (Nicholson et al., eds., 1998, Textbook of Influenza, Blackwell Science, Malden, Mass.; incorporated herein by reference). To assess the potency of the vaccine, the single radial immunodiffusion (SRD) test can be used (Schild et al., 1975, Bull. World Health Organ., 52:43-50 & 223-31; Mostow et al., 1975, J. Clin. Microbiol., 2:531; both of which are incorporated herein by reference).

Optionally, influenza virus for use in vaccines is grown in eggs, for example, in embryonated hen eggs, in which case the harvested material is allantoic fluid. Alternatively or additionally, influenza virus and/or the provided influenza hemagglutinin (HA) polypeptide may be produced from any method using tissue culture to grow the virus. Suitable cell substrates for growing the virus or otherwise recombinantly producing the engineered, structural influenza polypeptides include, for example, CHO cells, dog kidney cells such as MDCK or cells from a clone of MDCK, MDCK-like cells, monkey kidney cells such as AGMK cells including Vero cells, cultured epithelial cells as continuous cell lines, 293T cells, BK-21 cells, CV-1 cells, or any other mammalian cell type suitable for the production of influenza virus (including upper airway epithelial cells) for vaccine purposes, readily available from commercial sources (e.g., ATCC, Rockville, Md.). Suitable cell substrates also include human cells such as MRC-5 cells. Suitable cell substrates are not limited to cell lines; for example primary cells such as chicken embryo fibroblasts are also included.

Provided are compositions comprising one or more of the inactivated virus, virus-like particles or one or more of the HA polypeptides. Optionally, the inactivated virus or virus-like particles comprise an HA polypeptide with at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. Optionally, the HA polypeptide comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. Optionally, the compositions comprise a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 22nd Edition, Loyd V. Allen et al., editors, Pharmaceutical Press (2012). By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. If administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.

Optionally, a pharmaceutical composition can also contain a pharmaceutically acceptable carrier or adjuvant for administration of the vaccine, e.g., the HA polypeptides or VLPs. Optionally, the carrier is pharmaceutically acceptable for use in humans. The carrier or adjuvant should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, ammo acid copolymers and inactive virus particles.

An adjuvant refers to a substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as costimulatory molecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL. Optionally, the adjuvant is a squalene-based adjuvant. Squalene-based adjuvants are known and include, but are not limited to, MF59 and AS03.

Pharmaceutically acceptable carriers in therapeutic compositions can additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, can be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

The compositions of the presently disclosed subject matter can further comprise a carrier to facilitate composition preparation and administration. Any suitable delivery vehicle or carrier can be used, including but not limited to a microcapsule, for example a microsphere or a nanosphere (Manome et al. (1994) Cancer Res 54:5408-5413; Saltzman & Fung (1997) Adv Drug Deliv Rev 26:209-230), a glycosaminoglycan (U.S. Pat. No. 6,106,866), a fatty acid (U.S. Pat. No. 5,994,392), a fatty emulsion (U.S. Pat. No. 5,651,991), a lipid or lipid derivative (U.S. Pat. No. 5,786,387), collagen (U.S. Pat. No. 5,922,356), a polysaccharide or derivative thereof (U.S. Pat. No. 5,688,931), a nanosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle or conjugate (Goldman et al. (1997) Cancer Res 57:1447-1451 and U.S. Pat. Nos. 4,551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), and a polysome (U.S. Pat. No. 5,922,545).

Suitable formulations for the provided compositions can include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use. Some exemplary ingredients are SDS in the range of in some embodiments 0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/or mannitol or another sugar in the range of in some embodiments 10 to 100 mg/ml, in some embodiments about 30 mg/ml; and/or phosphate-buffered saline (PBS). Any other agents conventional in the art having regard to the type of formulation in question can be used. In some embodiments, the carrier is pharmaceutically acceptable. In some embodiments the carrier is pharmaceutically acceptable for use in humans.

Pharmaceutical compositions of the present disclosure can have a pH between 5.5 and 8.5, between 6 and 8, or about 7. Optionally, the pH can be maintained by the use of a buffer. The composition can be sterile and/or pyrogen free. The composition can be isotonic with respect to humans. Pharmaceutical compositions of the presently disclosed subject matter can be supplied in hermetically-sealed containers.

Pharmaceutical compositions can include an effective amount of one or more HA polypeptides and/or VLPs as described herein. Optionally, a pharmaceutical composition can comprise an amount that is sufficient to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction in physical symptoms. The precise effective amount for any particular subject will depend upon their size and health, the nature and extent of the condition, and therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation as practiced by one of ordinary skill in the art.

The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. Thus, the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges.

Provided herein is a method of eliciting an immune response to influenza virus in a subject, comprising administering the influenza HA polypeptide to the subject, wherein the administering elicits an immune response to influenza virus. Optionally, the method further includes administering an adjuvant to the subject. Optionally, the administering comprises administering to the subject a first influenza HA polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. Optionally, the method further includes administering to the subject a second influenza HA polypeptide having an amino acid sequence different from the first influenza HA polypeptide. Optionally, the first and second influenza HA polypeptides are administered simultaneously or concurrently. Optionally, the HA polypeptides are administered as inactivated virus or virus-like particles.

Also provided are methods of immunizing a subject against influenza virus, comprising administering to the subject a composition comprising one or more of the provided HA polypeptides, wherein administration immunizes the subject against influenza virus. Optionally, the HA polypeptides are administered as inactivated virus or virus-like particles. Optionally, the composition further comprises an adjuvant. Optionally, the composition is administered intramuscularly. Optionally, the composition comprises about 1 to about 25 μg of the VLP. Optionally, the composition comprises about 1 to 3 μg of the VLP. Optionally, the composition comprises about 3-15 μg of the VLP.

As used herein, vaccine refers to a preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of disease, such as an infectious disease. The immunogenic material may include, for example, attenuated or killed microorganisms (such as attenuated viruses), or antigenic proteins, peptides or DNA derived from them. Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous or intramuscular. Vaccines may be administered with an adjuvant to boost the immune response.

As used herein, an immune response refers to a response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or inflammation. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. In some embodiments, methods of measuring an immune response include hemagglutination inhibition (HAI) assays to assess functional antibody binding to the HA polypeptides described herein, thereby serving as surrogate measure of influenza vaccine efficacy. HAI assays may use chicken, turkey or horse erythrocytes for the detection of antibodies specific for H3N2. Protective immune responses can be demonstrated by eliciting an average HAI titer of greater than 1:40, which has been correlated with prevention and reduction of influenza illness. HAI antibody titers of approximately 1:32 to 1:40 will generally protect about 50% of subjects from infection after immunization with inactivated human influenza virus vaccine. See, e.g., Treanor, J. and Wright, P. F. Immune correlates of protection against influenza in the human challenge model. Dev. Biol. (Basel), 2003, 115:97-104, which is incorporated by reference herein in its entirety. Optionally, elicitation of a protective immune response can by identified by seroconversion rates. A protective level of seroconversion may be defined as at least a 4-fold rise in HAI titer, for example, a pre-administration or vaccination HAI titer of less than 1:10 and a post vaccinate titer of greater than or equal to 1:40. In other words, successful rates of seroconversion may be defined as the percentage of subjects with either a pre-vaccination HAI titer less than about 1:10 and a post-vaccination HAI titer of greater than about 1:40 or a pre-vaccination HAI titer greater than about 1:10 and a minimum four-fold rise in post-vaccination HAI antibody titer.

As used herein, an immunogen refers to a compound, composition, or substance which is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, as “immunogenic composition” is a composition comprising an immunogen (such as an HA polypeptide).

As used herein, the term “antigen” refers to a substance that prompts the generation of antibodies and can cause an immune response. It can be used interchangeably in the present disclosure with the term “immunogen”. In the strict sense, immunogens are those substances that elicit a response from the immune system, whereas antigens are defined as substances that bind to specific antibodies. An antigen or fragment thereof can be a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein can induce the production of antibodies (i.e., elicit the immune response), which bind specifically to the antigen (given regions or three-dimensional structures on the protein).

As used herein, immunize refers to rendering a subject protected from an infectious disease, such as by vaccination.

As used herein, administering a composition to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal and intramuscular.

Influenza HA polypeptides and VLPs comprising HA polypeptides, or compositions thereof, can be administered to a subject by any of the routes normally used for introducing recombinant virus into a subject. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation or oral. Parenteral administration, such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Administration can be systemic or local.

Influenza VLPs, or compositions thereof, are administered in any suitable manner, such as with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

Administration can be accomplished by single or multiple doses. The dose administered to a subject in the context of the present disclosure should be sufficient to induce a beneficial therapeutic response in a subject over time, or to inhibit or prevent influenza virus infection. The dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration. An appropriate dose can be determined by one of ordinary skill in the art using only routine experimentation.

The pharmaceutical compositions described herein can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical antibody pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and are adjusted depending on the particular therapeutic context or patient tolerance. The amount antibody adequate to accomplish this is defined as a “therapeutically effective dose.” The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest Remington's; Egleton, Peptides 18: 1431-1439, 1997; Langer, Science 249: 1527-1533, 1990.

“Therapeutically-effective amount” or “an amount effective to reduce or eliminate infection” or “an effective amount” refers to an amount of an antibody composition that is sufficient to prevent influenza virus infection or to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptoms associated with such an infection. It is not necessary that the administration of the composition eliminate the symptoms of influenza virus infection, as long as the benefits of administration of the composition outweigh the detriments. Likewise, the terms “treat” and “treating” in reference to influenza virus infection, as used herein, are not intended to mean that the subject is necessarily cured of infection or that all clinical signs thereof are eliminated, only that some alleviation or improvement in the condition of the subject is effected by administration of the composition.

As used herein, “treating” or “treatment of” a condition, disease or disorder or symptoms associated with a condition, disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently. In some embodiments, the terms treatment, treat or treating refer to methods of preventing the establishment of, or development of one or more consequences or symptoms associated with, infection by influenza A virus subtype H3N2, including fever, myalgia, headache, malaise, nonproductive cough, sore throat, rhinitis, weight loss, otitis media, nausea, vomiting, and death. For example, the terms treatment, treat, or treating can refer to methods of reducing the effects of one or more consequences or symptoms of infection by influenza A virus subtype H3N2. Thus, the methods of treatment disclosed herein can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more consequences or symptoms of infection by influenza A virus subtype H3N2, including fever, myalgia, headache, malaise, nonproductive cough, sore throat, rhinitis, weight loss, otitis media, nausea, vomiting, and death. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination. Optionally, the terms treatment, treat or treating refer to methods of shortening the duration of one or more symptoms of infection by influenza A virus subtype H3N2, including fever, myalgia, headache, malaise, nonproductive cough, sore throat, rhinitis, weight loss, otitis media, nausea, vomiting. In certain embodiments, the duration of symptoms is shortened to less than 2 weeks, to less than 7 days, and/or to less than 3 days.

Vertebrate,” “mammal,” “subject,” “mammalian subject,” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, cows, horses, goats, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as mice, sheep, dogs, cows, avian species, ducks, geese, pigs, chickens, amphibians, and reptiles.

A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. an autoimmune disease, inflammatory autoimmune disease, cancer, infectious disease, immune disease, or other disease) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophagelike synoviocytes, etc).

Also provided are kits comprising the HA polypeptides produced in accordance with the present disclosure which can be used, for instance, for therapeutic applications described above. Optionally, the kit comprises inactivated viruses of VLPs comprising the HA polypeptides. The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which includes an active agent that is effective for therapeutic applications, such as described above. The active agent in the composition can comprise the HA polypeptide or inactivated viruses or VLPs comprising the HA polypeptides. The label on the container indicates that the composition is used for a particular therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the claims.

EXAMPLE Example 1. Design and Characterization of 113 HA COBRA Influenza Vaccines

Antigen construction and synthesis. Influenza A HA nucleotide sequences isolated from human H3N2 infections were downloaded from the NCBI Influenza Virus Resource database (Bao et al., The Journal of Virology, 82(2):596-601 (2008)). Nucleotide sequences were translated into protein sequences using the standard genetic code. Full-length sequences from H3N2 viral infections isolated from human sources between 1968 and 2013 were used in this analysis. For each round of consensus generation, multiple alignment analysis was applied and the consensus sequence was generated using AlignX (Vector NTI). The final amino acid sequence, termed computationally optimized broadly reactive antigen (COBRA), was reverse translated and optimized for expression in mammalian cells, including codon usage and RNA optimization (Genewiz; Washington, D.C., USA). Fourteen H3N2 HA constructs were synthesized and inserted into the pTR600 expression vector, as previously described (Ross, et al., Nat Immunol 1:127-131 (2000)). COBRA HA antigens were designed to represent different antigenic space. Each COBRA HA structure was generated using the 3D-JIGSAW algorithm (Soding, J. Bioinformatics 21:951-960 (2005); and Soding, J., et al., Nucleic acids research 33:W244-248 (2005)) and renderings were performed using MacPyMol. A phylogenetic tree was inferred from hemagglutinin amino acid sequences using the maximum likelihood method and clade/sub-clade groupings were identified using Jalview (Dundee, UK).

In vitro expression. Human embryonic kidney (HEK) 293T cells (1×10⁶) were transiently transfected with 3 μg DNA expressing each COBRA or wild-type HA gene cassette. Cells were incubated for 72 hours at 37° C. and then lysed with 1% Triton-X 100 and clarified supernatant harvested following centrifugation. Cell lysates were then electrophoresed on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The blot was probed with pooled mouse antisera from infections with A/Brisbane/57/2007 and A/California/07/2009 viruses. HA-antibody complexes were then detected using goat anti-mouse IgG HRP (Southern Biotech; Birmingham, Ala., USA). HRP activity was detected using chemiluminescent substrate (Pierce Biotechnology; Rockford Ill., USA) and exposed to X-ray film (ThermoFisher; Pittsburgh, Pa., USA).

Functional characterization. To determine receptor-binding characteristics, virus-like particles (VLPs) were produced from transiently transfected with plasmids expressing HIV-1 Gag, (optimized for expression in mammalian cells), NA (A/mallard/Alberta/24/01; H7N3), optimized for expression in mammalian cells) and COBRA HA or HA from wild-type H3N2 strains and incubated for 72 hours at 37° C. (Medigen Inc, Rockville, Md., USA). Supernatants were collected and VLPs were purified via ultracentrifugation (100,000×g through 20% glycerol, weight per volume) for 4 h at 4° C. The pellets were subsequently resuspended in phosphate buffered saline PBS, pH 7.2 and stored at −80° C. until use. Protein concentration was determined by Micro BCATM Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, Ill., USA). COBRA HA VLPs were prepared in various amounts as measured by total HA protein and each individual preparation was two-fold serially diluted in v-bottom microtiter plates. An equal volume of 1% horse erythrocytes (RBC) (Lampire; Biologicals, Pipersville, Pa., USA) in PBS was added to the diluted VLPs and incubated for 60 minutes at room temperature. The HA titer was determined by the reciprocal dilution of the last well which contained agglutinated RBC.

Vaccine Preparation. HEK 293T cells were transiently transfected with plasmids expressing HIV-1 Gag, (optimized for expression in mammalian cells), NA (A/mallard/Alberta/24/01; H7N3), optimized for expression in mammalian cells) and COBRA HA or HA from wild-type H3N2 strains and incubated for 72 hours at 37° C. (Medigen Inc, Rockville, Md., USA). Supernatants were collected and cell debris removed by low speed centrifugation followed by vacuum filtration through a 0.22 μm sterile filter. VLPs were purified via ultracentrifugation (135,000×g through 20% glycerol, weight per volume) for 4 hours at 4° C. The pellets were subsequently resuspended in PBS pH 7.2 and stored in single use aliquots at −80° C. until use. Total protein concentration was determined by Micro BCATM Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, Ill., USA). Hemagglutination capacity was determined through serial dilution of VLPs, adding equal volume 0.8% turkey red blood cells to a V-bottom 96-well plate for a 30 min incubation at room temperature, and the highest dilution of VLP to have full agglutination of RBCs was considered the endpoint HA titer.

Quantification of HA content on 293T HA-NA-Gag VLPs was carried out using a high-affinity, 96-well flat bottom ELISA plate was coated with 5-10 μg of total protein of VLP and serial dilutions of a recombinant H3 antigen (3006_H3_Vc, Protein Sciences, Meriden, Conn.) in ELISA carbonate buffer (50 mM carbonate buffer, pH 9.5). The plate was incubated overnight at 4° C. on a rocker. Plates were then washed with PBS with 0.05% Tween-20 (PBST). Non-specific epitopes were blocked with 1% bovine serum albumin (BSA) in PBST solution for 1 hour at room temperature (RT). Buffer was removed then stalk-specific Group 2 antibody CR8020 (1:4000 dilution in blocking buffer) was added to plate and incubated for 1 hour at 37° C. Plates were washed and probed with goat anti-human IgG horseradish-peroxidase-conjugated secondary antibody (2040-05, Southern Biotech, Birmingham, Ala.) at dilution of 1:4000 for 1 hour at 37° C. Plates were washed then freshly prepared o-phenylenediamine dihydrochloride (OPD) (P8287, Sigma, City, State, USA) substrate in citrate buffer (P4922, Sigma) was added to wells, followed by 1N H2504 stopping reagent. Plates were read at 492 nm absorbance using a microplate reader (Powerwave XS, Biotek, Winooski, Vt.) and background was subtracted from negative wells. Linear regression standard curve analysis was performed using the known concentrations of recombinant standard antigen to estimate HA content in VLP lots. The results are shown in FIG. 1.

HA-specific content was determined by densitometry of reducing SDS-PAGE gels with the HA band identified by western blotting. Purified VLP's or other HA-containing samples and appropriate standards were electrophoresed on a 4-12% Bis-TRIS SDS-PAGE gel in MOPS buffer and transferred to a PVDF membrane (Life Technologies, Grand Island, N.Y.). The blot was probed with mouse monoclonal antibody 15B7 (Immune Technology Corporation, New York, N.Y.) and the HA-antibody complexes were detected by transilluminating fluorescence from a biotinylated goat anti-mouse IgG and the WesternDot™ 625 Western Blot Kit (Life Technologies). A gel run under identical conditions was stained with Sypro Ruby stain (Life Technologies). Gels and blots were imaged and the migration distances of all bands were recorded using a Chemi-Doc XRS+ camera system with QuantityOne software (Bio-Rad, Hercules Calif.). Molecular weights were calculated for all bands relative to MagicMarkXP standards (Life Technologies). The HA band was identified on the Sypro Ruby-stained gel as the one migrating at the same molecular weight for HA as determined from the western blot. Densities of HA bands from recombinant A/Puerto Rico/8/1934 HA standards (Immune Technology Corp.) were used to generate standard curves. The amount of HA in the purified VLP lanes was calculated by interpolation from the data of the standard curve. Experiments were performed in duplicate or triplicate and multiple exposure times were analyzed for all iterations.

Mouse studies. BALB/c mice (Mus musculus, females, 6-8 weeks) were purchased from Jackson Laboratories, (Bar Harbor, Me., USA) and housed in microisolator units and allowed free access to food and water and were cared for under USDA guidelines for laboratory animals and all procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Mice (16 mice per group) were vaccinated with purified VLPs (3.0 μg) based upon HA content from the densitometry assay or they were vaccinated with the monovalent inactivated split influenza vaccine (IIV), via intramuscular injection at week 0 and then boosted with the same vaccine at the same dose at weeks 4 and 8. In other cases, mice were primed with one VLP vaccine and boosted with a different vaccine. In addition, some mice were administered a cocktail of H3N2 COBRA VLP vaccines (1.5 ug dose of each of the two vaccines). Vaccines at each dose were formulated with AF03, an emulsified squalene-in-water adjuvant (Sanofi-Pasteur, Lyon, France). Final concentration after mixing 1:1 with VLPs is 2.5% squalene. AF03, an alternative squalene emulsion-based vaccine adjuvant prepared by a phase inversion temperature method or phosphate buffered saline alone was used as a mock vaccination. Twenty-eight days after each vaccination, blood was collected from anesthetized mice via the retro-orbital plexus and transferred to a microfuge tube. Tubes were centrifuged and serum samples were removed and frozen at −20±5° C.

Hemagglutination inhibition (HAI) assay. The HAI assay was used to assess functional antibodies to the HA able to inhibit agglutination of turkey erythrocytes. The protocol was adapted from the CDC laboratory-based influenza surveillance manual (Gillim-Ross, L., and K. Subbarao. Clin Microbiol Rev 19:614-636 (2006)). To inactivate non-specific inhibitors, sera were treated with receptor destroying enzyme (RDE; Denka Seiken, Co., Japan) prior to being tested (Bright, et al., Lancet 366:1175-1181 (2005); Bright, et al., Virology 308:270-278 (2003); Bright, et al., JAMA 295:891-894 (2006); Mitchell, et al., Vaccine 21:902-914 (2003); and Ross, et al., Nat Immunol 1:127-131 (2000)). Briefly, three parts RDE was added to one part sera and incubated overnight at 37° C. RDE was inactivated by incubation at 56° C. for approximately 30 minutes. RDE treated sera was two-fold serially diluted in v-bottom microtiter plates. An equal volume of each H3N2 virus, adjusted to approximately 8 HAU/50 μl, was added to each well. The plates were covered and incubated at room temperature for 20 min followed by the addition of 1% turkey erythrocytes (RBC) (Lampire Biologicals, Pipersville, Pa., USA) in PBS. Red blood cells were stored at 4° C. and used within 72 hours of preparation. The plates were mixed by agitation, covered, and the RBCs were allowed to settle for 1 hour at room temperature. The HAI titer was determined by the reciprocal dilution of the last well that contained nonagglutinated RBC. Positive and negative serum controls were included for each plate. All mice were negative (HAI≤1:10) for pre-existing antibodies to currently circulating human influenza viruses prior to vaccination.

Plaque Assay. Madin-Darby Canine Kidney (MDCK) cells were plated (5×10⁵) in each well of a 6-well plate. Samples were diluted (final dilution factors of 100 to 10⁻⁶) and overlayed onto the cells in 100 μl of DMEM supplemented with penicillin-streptomycin and incubated for 1 hour. Samples were removed, cells were washed twice and media was replaced with 2 ml of L15 medium plus 0.8% agarose (Cambrex; East Rutherford, N.J., USA) and incubated for 72 hours at 37° C. with 5% CO2. Agarose was removed and discarded. Cells were fixed with 10% buffered formalin, and then stained with 1% crystal violet for 15 minutes. Following thorough washing in dH2O to remove excess crystal violet, plates were allowed to dry, plaques counted, and the plaque forming units (PFU)/ml were calculated.

Statistical Analysis. Statistical significance of the antibody data was determined using a two-way analysis of variance (ANOVA) with Bonferroni's post-test to analyze differences between each vaccine group for the different test antigens (multiparametric). Differences in weight loss, sickness score, and viral titers were analyzed by two-way ANOVA, followed by Bonferroni's post-test for each vaccine group at multiple time points. Significance was defined as p<0.05. Statistical analyses were done using GraphPad Prism software.

FIG. 2 is a schematic representation of HAI results in table format. Following vaccination in mice, serum samples were collected at week 10 post-vaccination and sera was tested in an HAI assay against a panel of wild-type H3N2 influenza viruses. Each number represents the number of mice that have a positive HAI titer of 1:40 or greater. The number of mice (out of 5 mice per group) that reached the 1:40 threshold is listed as the number in the table.

Example 2. Analysis and Characterization of 113 HA COBRA Vaccines

Materials and Methods.

Antigen construction and synthesis. Influenza A HA protein sequences from 6,430 human H3N2 infections collected from Jan. 1, 1968-Dec. 15, 2013 were downloaded from the Global Initiative on Sharing Avian Influenza Data (GISAID) database and organized by the year of collection. For each round of consensus generation, multiple sequence alignment was performed using Geneious® MUSCLE alignment method and phylogenetic neighbor-joining trees were constructed, such that trees were rooted to the oldest sequences collected in 1968. Full-length HA sequences were aligned and the most common residue found among a designated set of viruses were used to yield the primary consensus sequence and ambiguities were avoided through alignment of odd-number of sequences. Multiple rounds of consensus assembly were layered to yield secondary, tertiary, and quaternary consensus sequences that were designed to represent different antigenic spaces that overlapped periods of time that wild-type vaccine strains that were recommended from 1968-2013. The final amino acid sequences were then renamed T-series computationally optimized broadly reactive antigen (COBRA). Fifteen H3N2 HA constructs were synthesized and inserted into the pTR600 expression vector, as previously described (Ross et al., Nat. Immunol. 1:127-131 (2000)). COBRA HA antigens were designed to represent different antigenic spaces. For example, constructs T-2 through T-7 were designed to represent non-overlapping periods of time spanning 4-6 years using H3 sequences from human isolates collected between 1980 through 2010, and T-11 was generated using HA sequences isolated from 2011-2013. The COBRA HA constructs, T-9, T-12, T-13, T-14, T-15, T-16, and T-17, represent an antigenic space from 1968-2013 using various multi-consensus layering approaches.

Cartography and predictive structure-homology modeling methods. Human, influenza A (subtype H3N2) hemagglutinin protein sequences were obtained from the NCBI Influenza Virus Resource database, trimmed to remove signal peptides, transmembrane regions, and cytoplasmic tails, and the resulting ectodomain sequences were aligned using MAFFT. The pairwise dissimilarity matrix was calculated from the multiple-sequence alignment based on the Hamming distance between pairs of sequences with no prior assumptions regarding the function or structure of the sequences. Principal component analysis (PCA) was applied to the dissimilarity matrix for the purpose of dimension reduction and to facilitate visualization of the relative distances between HA proteins. The first two or three principal components were retained for visualizing protein relationships in sequence space and represent a reasonable approximation of the general structure of the phylogenetic tree. Calculations were performed using custom scripts written in python and R. Each COBRA HA predictive structure was generated using the SWISS-MODEL algorithm (Kiefer et al., Nucleic Acids Res. 37:D387-392 (2009); Arnold et al., Bioinformatics 22:195-201 (2006); and Biasini et al., Nucleic Acids Res. 42:W252-258 (2014)) implementing the PDB ID 2yp2.1.a, the crystal structure of a human H3N2 2004 virus (Lin et al., PNAS 109:21474-21479 (2012), as the HA template and renderings were performed using MacPyMol. Additional modifications were performed by color-coding of antigenic sites based on literature (Koel et al., Science 342:978-979 (2013); Lewis et al., Journal of Virology 88:4752-4763 (2014); and Stray and Pittman, Virol. J. 9:91 (2012)). Separate frames were collected of the surface-rendered hemagglutinin structures at the matched x-, y-, and z-coordinates.

Vaccine preparation. Mammalian 293T cells were transfected with one of three plasmids expressing either the influenza neuraminidase (A/mallard/Alberta/24/01, H7N3), the HIV p55 Gag sequences and one of the the various H3N2 wild-type or COBRA HA expressing plasmids on previously described mammalian expression vectors (Green et al., Journal of Virology 77:2046-2055 (2003)). Following 72 hours of incubation at 37° C., supernatants from transiently transfected cells were collected, centrifuged to remove cellular debris, and filtered through a 0.22 μm pore membrane. Mammalian virus-like particles (VLPs) were purified and sedimented by ultracentrifugation on a 20% glycerol cushion at 135,000×g for 4 hours at 4° C. VLPs were resuspended in phosphate buffered saline (PBS) and total protein concentration assessed by conventional bicinchoninic acid assay (BCA). Hemagglutination activity of each preparation of VLPs was determined by adding equal volume turkey red blood cells (RBCs) to a V-bottom 96-well plate and incubating with serially diluted volumes of VLPs for a 30 min incubation at RT. The highest dilution of VLP with full agglutination of RBCs was considered the endpoint HA titer.

Deterimination of HA content. A high-affinity, 96-well flat bottom ELISA plate was coated with 5-10 μg of total protein of VLP and serial dilutions of a recombinant H3 antigen (3006_H3_Vc, Protein Sciences, Meriden, Conn.) in ELISA carbonate buffer (50 mM carbonate buffer, pH 9.5) and plate was incubated overnight at 4° C. on a rocker. The next morning, plates were washed PBS with 0.05% Tween-20 (PBST), then non-specific epitopes were blocked with 1% bovine serum albumin (BSA) in PBST solution for 1 hour at room temperature (RT). Buffer was removed then stalk-specific Group 2 antibody CR8020 was added to plate and incubated for 1 hour at 37° C. Plates were washed and probed with goat anti-human IgG horseradish-peroxidase-conjugated secondary antibody (2040-05, Southern Biotech, Birmingham, Ala.) for 1 hour at 37° C. Plates were washed then freshly prepared o-phenylenediamine dihydrochloride (OPD) (P8287, Sigma, City, State, USA) substrate in citrate buffer (P4922, Sigma, St. Louis, Mo.) was added to wells, followed by 1N H2SO4 stopping reagent. Plates were read at 492 nm absorbance using a microplate reader (Powerwave XS, Biotek, Winooski, Vt.) and background was subtracted from negative wells. Linear regression standard curve analysis was performed using the known concentrations of recombinant standard antigen to estimate HA content in VLP lots.

Mouse studies. BALB/c mice (Mus musculus, females, 6 to 8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, Me., USA) and housed in microisolator units and allowed free access to food and water and were cared for under USDA guidelines for laboratory animals. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Mice (5 mice per group) were vaccinated with purified VLPs (3.0 μg/mouse) based upon HA content from the ELISA quantification and vaccines were delivered via intramuscular injection at week 0 and then boosted with the same vaccine at the same dose at weeks 4 and 8. In other cases, mice were primed with one VLP vaccine and boosted with a different vaccine. In addition, some mice were administered a cocktail of H3 COBRA VLP vaccines (1.5-μg dose of each of the two vaccines) or phosphate-buffered saline alone as a mock vaccination. Vaccines at each dose were formulated with an emulsified squalene-in-water adjuvant (Sanofi Pasteur, Lyon, France). The final concentration after mixing 1:1 with VLPs is 2.5% squalene. Twenty-eight days after each vaccination, blood samples were collected via the submandibular cheek and transferred to a microcentrifuge tube. The tubes were centrifuged, and serum samples were removed and frozen at −20° C.±5° C.

HAI assay. The hemagglutination inhibition (HAI) assay was used to assess functional antibodies to the HA able to inhibit agglutination of guinea pig and as well as turkey erythrocytes. The protocols were adapted from the WHO laboratory influenza surveillance manual (Organization, W. H. & Network, W. G. I. S. Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza. (World Health Organization, 2011)) and uses the host-species that is frequently used to characterize contemporary H3N2 strains that have preferential binding to alpha (2,6) linked sialic acid receptors (Katz, et al., Expert Rev. Anti. Infect. Ther. 9:669-683 (2011); Oh et al., Journal of Clinical Microbiology 46:2189-2194 (2008)). It was also compared with turkey erythrocytes to compare whether there was a differential in HAI depending on erythrocyte used. To inactivate nonspecific inhibitors, sera were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Co., Japan) prior to being tested. Briefly, three parts of RDE was added to one part of sera and incubated overnight at 37° C. RDE was inactivated by incubation at 56° C. for ˜30 min. RDE-treated sera were diluted in a series of twofold serial dilutions in in v-bottom microtiter plates. An equal volume of each H3N2 virus, adjusted to approximately 8 hemagglutination units (HAU)/50 μl, was added to each well. The plates were covered and incubated at room temperature for 20 min, and then 0.8% guinea pig erythrocytes (Lampire Biologicals, Pipersville, Pa., USA) in PBS were added. Red blood cells were stored at 4° C. and used within 72 hours of preparation. The plates were mixed by agitation and covered, and the RBCs were allowed to settle for 1 hour at room temperature. The HAI titer was determined by the reciprocal dilution of the last well that contained nonagglutinated RBCs. Positive and negative serum controls were included for each plate. All mice were negative (HAI≤1:10) for preexisting antibodies to currently circulating human influenza viruses prior to vaccination and seroprotection was defined as HAI titer>1:40 and seroconversion as a 4-fold increase in titer compared to baseline, as per the WHO and European Committee for Medicinal Products to evaluate influenza vaccines (Agency, E. M. in EMA/CHMP/VWP/457259/2014 (ed Committee for Medicinal Products for Human Use) (London E14 4HB, UK, 2014)); however, often examined was a more stringent threshold of >1:80. Mice are naïve and seronegative at the time of vaccination, thus seroconversion and seroprotection rates are interchangeable in this study.

Viruses. H3N2 viruses were obtained through the Influenza Reagents Resource (IRR), BEI Resources, the Centers for Disease Control (CDC), or provided by Sanofi-Pasteur. Viruses were passaged once in the same growth conditions as they were received, in either embryonated chicken eggs or semi-confluent Madin-Darby canine kidney (MDCK) cell culture as per the instructions provided the WHO9. Virus lots were tittered with both guinea pig and turkey erythrocytes and made into aliquots for single-use applications. The H3N2 vaccine panel includes the following strains: A/Hong Kong/1/1968 (NR-28620), A/Port Chalmers/1/1973, A/Mississippi/1/1985×PR/8 (NR-3502), A/Sichuan/60/1989×PR/8 (NR-3492), A/Nanching/933/1995, A/Sydney/05/1997, A/Panama/2007/1999, A/Fujian/411/2002, A/New York/55/2004 (FR-462), A/Wisconsin/67/2005 (FR-397), A/Brisbane/10/2007, A/Perth/16/2009, ANictoria/361/2011, A/Texas/50/2012, and A/Switzerland/9715293/2013 (J1506B), A/Hong Kong/4801/2014. All viruses were characterized for ability to agglutinate turkey and guinea pig erythrocytes.

The Results are shown in FIGS. 3-6. FIG. 3 are graphs showing H3 COBRA vaccines have enhanced reactivity to H3N2 vaccine panel. T-6 COBRA elicits reactivity to all three Nan/95, Pan/99 and Fuj/02 while no wild type (WT) virus cross reacts against all three with greater seroconversion. T-7 COBRA elicits reactivity to Wis/05, Bris/07 and Perth/09 while WT vaccines lose cross reactivity to each other. FIG. 4 is a schematic showing the H3 COBRA elicited antisera react against more antigenically diverse antigens by HAI than conventional strains. FIG. 5 is a schematic showing cross reactivity of T-6, T-7, T-8, T-10 and T-11 COBRA vaccines. FIG. 6 is a graph showing the strains with HAI reactivity against a panel of 15 2004-2009 virus variants.

As described herein, unique H3 COBRA constructs can be designed against antigenic spaces by time. The wild type (WT) and COBRA HA expressing VLPs were tested in mice and the data collectively shows the H3 COBRA vaccines have enhanced reactivity. Specifically, H3 COBRA VLPs elicited antisera reacts against more antigenically diverse H3N2 stains as determined by HAI than WT VLPs. Further, broad HAI breadth was obtained using heterologous prime-boost immunization. Finally, T-7 elicited superior HAI reactivity and T-8 and T-11 elicited high reactivity to a panel of 15 2004-2009 variants as compared to the WT vaccines. 

What is claimed is:
 1. A recombinant influenza HA polypeptide comprising at least 95% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.
 2. A recombinant influenza HA polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.
 3. A recombinant influenza HA polypeptide comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.
 4. A composition comprising the influenza HA polypeptide of any one of claim 1-3.
 5. A method of eliciting an immune response to influenza virus in a subject, comprising administering the influenza HA polypeptide of any one of claims 1-3, wherein the administering elicits an immune response to influenza virus.
 6. The method of claim 5, further comprising administering an adjuvant to the subject.
 7. The method of claim 5 or 6, wherein the administering comprises administering to the subject a first influenza HA polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, and wherein the method further comprising administering to the subject a second influenza HA polypeptide having an amino acid sequence different from the first influenza HA polypeptide.
 8. The method of claim 7, wherein the first and second influenza HA polypeptides are administered simultaneously or concurrently.
 9. A virus-like particle comprising the polypeptide of any one of claims 1-3.
 10. The virus-like particle of claim 9, further comprising an influenza neuraminidase (NA) polypeptide and an influenza matrix (M1) polypeptide.
 11. The virus-like particle of claim 10, wherein the wherein the amino acid sequence of the influenza NA polypeptide is at least 95% identical to SEQ ID NO:27, the amino acid sequence of the influenza M1 polypeptide is at least 95% identical to SEQ ID NO:29, or both.
 12. The virus-like particle of claim 11, further comprising an influenza neuraminidase (NA) polypeptide and an HIV GAG polypeptide.
 13. A composition comprising the virus-like particles of any one of claims 9-12, and a pharmaceutically acceptable excipient.
 14. A method of immunizing a subject against influenza virus, comprising administering to the subject the composition of claim 13, wherein administration immunizes the subject against influenza virus.
 15. The method of claim 14, wherein the composition further comprises an adjuvant.
 16. The method of claim 14, wherein the composition is administered intramuscularly.
 17. The method of any one of claims 14-16, wherein the composition comprises about 1 to about 25 μg of the VLP.
 18. The method of any one of claims 14-16, wherein the composition comprises about 1 to 3 μg of the VLP.
 19. The method of any one of claims 14-16, wherein the composition comprises about 3-15 μg of the VLP.
 20. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding an influenza hemagglutinin (HA) polypeptide, wherein the nucleotide sequence encoding the HA polypeptide is at least at least 95% identical to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.
 21. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding an influenza hemagglutinin (HA) polypeptide, wherein the nucleic acid sequence comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.
 22. A vector comprising one or more of the nucleic acid sequences of claim 20 or
 21. 23. A vector comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.
 24. A plurality of vectors comprising (i) a vector comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, encoding an HA polypeptide; (ii) a vector comprising a nucleic acid sequence encoding an influenza neuraminidase (NA); and (iii) a vector comprising a nucleic acid sequence encoding an HIV GAG polypeptide.
 25. A plurality of vectors comprising (i) a vector comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, encoding an HA polypeptide; (ii) a vector comprising a nucleic acid sequence encoding an influenza neuraminidase (NA); and (iii) a vector comprising a nucleic acid sequence encoding an influenza M1 polypeptide.
 26. A cell comprising the vector of claim 22 or 23 or the plurality of vectors of claim 24 or
 25. 27. A method of making a virus like particle (VLP) comprising culturing the cell of claim 26 under conditions to produce the VLP and isolating the VLP.
 28. A method of making a VLP comprising transfecting a host cell with a vector encoding the HA polypeptide of any one of claims 1-3, a vector encoding an influenza NA polypeptide and a vector encoding an influenza M1 polypeptide under conditions sufficient to allow for expression of the HA, Ml and NA polypeptides and isolating the VLP.
 29. A method of making a VLP comprising transfecting a host cell with a vector encoding the HA polypeptide of any one of claims 1-3, a vector encoding an influenza NA polypeptide and a vector encoding a HIV GAG polypeptide under conditions sufficient to allow for expression of the HA, HIV GAG and NA polypeptides and isolating the VLP. 